There have been numerous attempts to produce high levels of a desired amino acid sequence by introducing a foreign (heterologous) nucleic acid into host cells and then expressing that nucleic acid inside the cells. Eukaryotic cells and especially mammalian cells have been employed with mixed results. For example, despite much effort toward improving methods of making recombinant mammalian cells that produce high levels of the amino acid sequence, most of the cells do not express the nucleic acid at sufficient levels. Thus, there is a need in the field to have methods for generating recombinant mammalian cells that express the introduced nucleic acid at high levels.
In general, two methods have been used to increase expression of heterologous nucleic acid in mammalian cells. One approach has been to enhance nucleic acid copy number by cell selection techniques such as drug resistance amplification. Another approach has been to increase expression of the nucleic acid inside the cells, e.g., by recombinantly adding one or more a beneficial control elements to that nucleic acid. See generally Kaufman, R. J. and P. A. Sharp (1982) J Mol. Biol. 159: 601; Sambrook et al. Molecular Cloning (2d ed. 1989) and Ausubel et al. Current Protocols in Molecular Biology, (19890 John Wiley & Sons, New York.
In particular, drug resistance amplification has been reported to involve cell transformation with two genes, one of which encodes an amino acid sequence of interest, such as a heterologous protein, and the other which encodes a selectable gene marker such as dihydrofolate reductase (DHFR). In instances in which the gene marker is DHFR, transformed cells are cultured in the presence of the selecting drug methotrexate (MXT). The normally cytotoxic effects of MTX are substantially eliminated by expression of the DHFR. Transformed cells survive because they have increased (amplified) DHFR copy number to a sufficiently high level. Nucleic acid sequence encoding the desired amino acid sequence is also amplified, thereby boosting expression of that sequence. See e.g., Kaufman, R. J and P. A. Sharp, supra.
However, drug resistance amplification and related techniques have recognized drawbacks. For example, generation of most recombinant mammalian cell lines using the technique is time-consuming and may require several months to perform. Additionally, there has been recognition that when the heterologous nucleic acid is amplified, standard nucleic acid sequencing methods can be negatively impacted. Further, it is can be quite difficult to maintain sufficient nucleic acid copy number without imposing severe and sometimes long-term selection pressure. A cell selection strategy calling for extended selection pressure can be expensive and may present regulatory issues such as when a heterologous protein is being produced for pharmaceutical use.
In addition, drug resistance amplification may not be able to enhance expression of specific heterologous nucleic acids to levels sufficient for applications such as commercial or research use.
Further problems with drug resistance amplification include genetic instability of cloned cell lines. These problems are highly significant. For example, the method often produces recombinant cells that evolve from unstable gene amplification events, e.g., formation of double minute chromosomes. These cells can lose desired characteristics when the selecting drug is removed. See Kaufman, R. J. et al. (1985) Mol. Cell. Biol. 5: 1750 and references cited therein.
Additionally, optimal practice of most drug resistance amplification techniques has required use of highly specialized cells, vectors and/or cell growth conditions. For example, most of the methods employ host cells that have been genetically manipulated in specific ways. Such mutant cells may not be suited for some applications. As an illustration of the difficulties, drug resistance amplification involving DHFR and MTX will not work optimally in cells carrying a normal DHFR gene. Disabling that normal gene can be a lengthy and laborious process. See e.g., Wigler et al. (1980) PNAS (USA) 3567; and Urlaub and Chasin, (1980) PNAS (USA) 77: 4216.
More particular drawbacks of DHFR/MTX amplification methods relating to use of specific vectors and growth conditions have been disclosed. See e.g., Kaufman and Sharp, supra; Schimke, R. Cell (1984) 37:705. See also U.S. Pat. Nos. 5, 686,263; 4,956,288 and 5,585,237 and references cited therein.
It would be useful to have methods for making recombinant cells that are flexible and can be used to produce recombinant cell lines that are genetically stable. It would be particularly desirable to have methods for producing recombinant mammalian cell lines that significantly reduce or avoid use of highly specialized cells, vectors and/or growth conditions. It would be further desirable to have recombinant mammalian cell lines made by those methods that produce high levels of an amino acid sequence of interest such as a heterologous protein.